Bioreactor for fermenting solids

ABSTRACT

The invention relates to bioreactor for fermenting solid substrates, comprising a fermentation vessel ( 2 ), a device for feeding bioreactive substances and a nozzle arrangement in the fermentation vessel ( 2 ). The invention is characterized in that a nozzle arrangement ( 10, 20 ) consisting of a plurality of pipes ( 14, 24 ) which project into the reaction chamber ( 49 ) of the fermentation vessel ( 2 ) in parallel and which are provided with nozzles ( 16, 28 ) is situated in the fermentation vessel ( 2 ). The invention also relates to a method for aerobically fermenting solids. A reaction medium containing these solids is mixed using a compressed gas ( 48 ) which is guided into the reaction mixture from above.

This application is a 371 of PCT/EP00/08929 filed on Sep. 13, 2000.

The present invention relates to a bioreactor for fermenting solids, anda corresponding fermentation method.

The conversion of solid, water-insoluble or particular substances infermenters involves a wide variety of problems, primarily relating toaeration, mixing and the addition of nutrient media. In addition, if thesubstrate to be converted is to be synergistically attacked by a numberof different microorganisms, it Is required to selectively supply thereaction space with nutrients and oxygen. In large-volume reactors, suchfermentations currently cannot be realized due to the complicated mixingand the resulting defective aeration and deficient supply of substrates.

In commercially available reactor systems, the mixing is effected bymechanical agitating systems. In addition, U.S. Pat. No. 4,846,964describes a fluidized-bed bioreactor system for converting coal tomicrobiologically liquefied coal products in which an upflowing aqueousstream keeps the coal particles fluidized. The above mixing methods havea drawback in that sufficient mixing and thus a high substrate turnoveris no longer possible in the fermentation of higher substrateconcentrations or substrates which tend to agglomerate.

Now, it has been the object of the present invention to provide abioreactor and a fermentation method which overcome the drawbacks of theprior art and, in particular, ensure a sufficient mixing of thesubstrate to be fermented.

Surprisingly, it has now been found that a sufficient mixing of thereaction medium, which contains solid or water-insoluble fermentationsubstrates, can be achieved in a bioreactor by purposefully introducinga compressed gas continuously or in compressed gas pulses.

The above object is achieved by a bioreactor having the features asdescribed below, and a method for aerobic fermentation having thefeatures as described below.

The bioreactor serves for the fermentation of water-insoluble orparticular substrates, such as wood (which can be degraded only byparticular microorganisms due to its lignin content), coals (with thegoal of using the liquid fermentation products as starting materials forthe chemical industry or for thermal utilization), for the remediationof soils loaded with xenobiotics, for the rapid fermentation of organicwaste products, for biological waste water purification, and for thepretreatment of basic materials of the chemical industry.

The proposed bioreactor for the first time permits the optimum aerationand mixing of solid, water-insoluble and particular substances, which isnecessary for microorganisms, by using at least one specific nozzlearrangement which is introduced into the substrate to be fermented andpneumatically supplied with compressed gas. Both the supply of oxygen tothe microorganisms and the supply thereto of nutrient media,cosubstrates, vitamins, minerals, buffers or antibiotics are effected bya single pneumatic pressure system. In a slightly modified form, thebioreactor can be used In any size from a five-liter laboratory scale toan industrial, multi-hectoliter scale. With simple modifications, theproposed bioreactor can also be used as a conventional liquid/solidphase, solid phase, falling film, fed batch or air-lift reactor.

According to the invention, a first, vertically extending nozzlearrangement can be extended into and retracted from the reaction spaceof the fermentation vessel. Thus, the nozzle arrangement can be shiftedin a vertical direction to enable movement of the nozzle arrangementwhile pressurized with compressed gas, for a better mixing and aeration.However, if the fermentation process is to proceed under sterileconditions, it is imperative that the reaction space be kept closed. Thenozzle arrangement consists of pipes which vertically protrude into thefermentation vessel and are provided with nozzles on their lower ends.In this way, the compressed gas or the liquid bioreactive substrate canbe introduced near the bottom of the fermentation vessel. The nozzlearrangement is also suitable for penetrating granular solids present inthe fermentation broth. The vertical pipes can have different lengthsand can be exchanged. Further, the nozzle arrangement, when retractedfrom the fermentation vessel, can be cleaned in a simple way.

In addition, a second horizontal nozzle arrangement can be providedwhich consists of interconnected pipes horizontally extending inparallel through the reaction space. The pipes have nozzles distributedon their coat surface.

The horizontal nozzle arrangement can be used for mixing additionally tothe vertical nozzle arrangement.

If the horizontal nozzle arrangement is to rotate for a better mixing ofthe fermentation substrate, the vertical arrangement of nozzles must bemoved upwards, or the vertical pipes must be selected so as not tohinder the rotating of the horizontal nozzle arrangement, i.e., the“normal” pipes must be replaced by shorter pipes.

An advantageous further development of the bioreactor according to theinvention has a measuring device in which several measuring electrodes,for example, are provided in a measuring chamber for measuring a mediumremoved from the fermentation vessel. The measuring chamber is connectedwith the fermentation vessel through a feed line in order to feed mediumto be measured from the fermentation vessel into the measuring chamber.To recirculate the medium into the fermentation vessel after measuring,the measuring chamber is further connected with the fermentation vesselthrough a recirculating line. The special about this arrangement is thefact that a pressure chamber is inserted upstream from the measuringchamber, where a defined pressure can build which corresponds to thedisplaced volume in the measuring chamber. According to the invention,the recirculation of the measured medium is effected by applyingpressure to the measuring chamber so that the medium is pressed backinto the fermentation vessel.

In known bioreactors, measurements are usually performed directly withinthe fermentation vessel. Since solid substances are contained in thefermentation vessel, the measuring electrodes are often damaged. Forexternal measurement, in known bioreactors, the medium to be measured istransported into a measuring chamber by peristaltic pumps. Such pumpsare subject to high wear and are not suitable for transporting largervolumes.

Because the medium is transported by pressure according to theinvention, such pumps which are subject to wear and thus to intensivemaintenance can be dispensed with. Further, it is possible to transportlarge volumes. Another advantage of transport by pressure is the factthat the sterility is not affected. This means that no foreign mattergets into the fermentation vessel during the pumping process.

The feeding of medium through the feed line into the measuring chambercan be effected by negative pressure. The measuring chamber according tothe invention can further be used as an additional recirculation system.Such a recirculation system can perform an additional aeration of thesubstrates present in the fermentation vessel.

Further advantageous embodiments of the bioreactor can be seen from thefurther dependent claims.

In the method according to the invention, for the aerobic fermentationof solid substrates, a reaction medium containing such solid substratesis thoroughly mixed by compressed gas introduced from above into thereaction medium. This thorough mixing dramatically increases thefermentation rate. According to the present invention, “to introducefrom above into the reaction medium” means that compressed gas isintroduced into the reaction medium by means of suitable devices (suchas pipes provided with nozzles) which extend into the reaction mediumfrom above. The length of the pipes can be selected to reach below thesurface of the reaction medium at any level in the introduced workingcondition (i.e., with or without introduced compressed gas).

In the following, the invention will be further described by the Figuresand Examples, wherein:

FIG. 1 shows a bioreactor having a pressure vessel for bioreactivesubstances and for compressed air;

FIG. 2 shows a frontal view of the bioreactor;

FIG. 3 shows a side view of the bioreactor;

FIG. 4 shows a cross-sectional view through the fermentation vessel ofthe bioreactor;

FIG. 5 shows a cross-sectional view along line V—V in FIG. 4;

FIG. 6 shows a media mixing and compressed air feeding arrangement forthe bioreactor;

FIG. 7 is a schematic view of a measuring means connected with thefermentation vessel; and

FIG. 8 shows the course of reaction of the fermentation charge describedin Example 1.

The bioreactor shown In FIG. 1 for the fermentation of solid substratesand for performing one of the fermentation processes described in moredetail below has a fermentation vessel 2 which can be hermeticallysealed with a pressure lid 8.

Fermentation vessel 2 is provided with at least one nozzle arrangement10, 20 extending into the reactor space 49 to which compressed air 48 issupplied through a pressure vessel 44, through a compressed gas line 3for a vertical nozzle arrangement 10 and through a compressed gas line 4for a horizontal nozzle arrangement 20.

The pressure vessel 44 additionally contains a bioreactive liquidsubstance 50 which can be supplied through a stop valve 7 and a pressureline 5 to the horizontal nozzle arrangement 20, and/or through apressure line 9 to the vertical nozzle arrangement 10.

Consequently, the vertical nozzle arrangement 10 and/or the horizontalnozzle arrangement 20 may also be used as a supply means for bioreactivesubstances as an alternative of pressurizing with compressed gas.

On the bottom of the pressure vessel 44 is provided a discharge line 11with a stop valve 12. On the top end of the pressure vessel 44 isprovided a feed line 13 for supplying bioreactive substances 50 and astop valve 15.

The compressed gas 48, e.g., compressed air, is supplied through a feedline 17 and a stop valve 18.

Juxtaposed to the pressure vessel 44 is a rising pipe 19.

The fermentation 2 further has a pressure compensating means 21 with astop valve. On the bottom of the fermentation vessel 2, a drainingchannel 36 covered by a wire mesh 38 is provided below a conical bottomsection 32 (FIG. 2), so that liquid contained in the fermentation vessel2 is coarse-filtered prior to being discharged into the drainingchannel. The bottom of the draining channel 36 is inclined fromhorizontal, a draining valve 40 being provided at the front side 51 ofthe fermentation vessel at the lowest position of the draining channel36.

On the front side 51 of the fermentation vessel 2 is provided aremovable door 53 to enable cleaning of the interior space and of thewire mesh 38 without having to open the cover plate 8 sealing thereactor space 49. On the draining channel 36 are provided two windows 41facing each other, which enable determination of the optical density ofthe fermented substance with an optical sensor (FIG. 3). Additionalwindows 62, 64 provided on the lateral walls enable visual control ofthe fermentation conditions, but these are not obligatory.

Instead of or in addition to a subsequently described external measuringchamber 72, on one side of the fermentation vessel 2, there may beprovided a sealable means 66 for measuring electrodes (e.g., pH, O₂partial pressure, conductivity, ion specificity etc.) and a pressurecompensating means 21 for the controlled exhausting of air or gasesformed during fermentation from the fermentation vessel 2.

All components which will come into contact with medium or gases can beindependently autoclaved.

The bioreactor according to the invention can further have devices forheat exchange, i.e., heating and cooling. Thus, the fermentation vessel2 can have a double wall to form a cavity 51 a. This cavity is formedbetween the inner and outer walls of vessel 2. The cavity 51 a isconnected, for example, with a correspondingly temperature-controlledheat exchange fluid reservoir (e.g., water or oil reservoir) through aconnecting pipe 51 b and discharge pipe 51 c. The water or oil which hasa defined temperature due to an external thermostat flows around vessel2. Due to this device, the bioreactor can be operated with steplesscontrol in a temperature range of from 10 to 90° C., whereby, on the onehand, the optimum temperature for the different microorganisms can beadjusted in the bioreactor, and on the other hand, the whole bioreactorincluding its contents can be mildly sterilized or tyndallized.Therefore, such a sterilization/tyndallization of a whole bioreactor inan integrated system, which has not been previously described as such,has significant advantages in the application of the system.

As can be seen from FIGS. 2 and 3, the fermentation vessel 2 is held bya frame 6. The frame 6 also accommodates two spindles 22 by means ofwhich the pressure lid 8 of fermentation vessel 2 can be lowered ontothe latter or lifted. The pressure lid 8 at the same time forms thevertical nozzle arrangement by effecting the supply of compressed gas orbioreactive substances through the pressure lid 8 to the vertical pipes14 extending vertically from the pressure lid 8. The vertical pipes 14can be of different lengths, the tips of the pipes 14 with nozzles 16being positioned in the bottom portion of the fermentation vessel whenthe pressure lid 8 is closed, By lowering the pressure lid, the nozzles16 can also penetrate solid substrates present in the reactor space. Inthe closed position of the pressure lid, the fermentation vessel 2 canbe hermetically sealed.

The spindles 22 are actuated by means of crank handles 27 provided onthe top of frame 6.

A hollow pressure plate 8 serves as the pressure lid of bioreactor 1,being one of two possibilities for aerating the bioreactor space in thefermentation vessel 2 or supplying it with media. It can be verticallyguided by rails and hermetically seals the fermentation vessel 2 duringoperation. When the bioreactor 1 is to be charged with the substrate tobe converted, the pressure lid 8 can also be lifted hydraulically orpneumatically in addition to the mechanical way described above.

On its top side, pressure lid 8 has a manually or electronicallycontrollable port for supplying compressed air (or defined gases).Optionally, the port may also be used for the supply of liquid definedmedia, e.g., required cosubstrates, vitamins, minerals, buffers orantibiotics. Within pressure lid 8, the air first impinges on a baffle45. On the lower side of pressure lid 8, exchangeable pipes 14 withnozzles 16 are screwed into threads. When pressure builds in thepressure lid 8, the air continuously flows through the nozzles 16provided at the tips of pipes 14 into the reactor space 49 offermentation vessel 2, aerating it uniformly. In addition to the mildmixing of the reactor space by the continuous flow of air, a thoroughmixing of the reactor space can optionally be effected by strong pulsesof compressed air applied in defined intervals.

Due to the different lengths of the exchangeable pipes 14 which can bescrewed into the pressure lid 8, it is possible to aerate the reactorspace 49 in a well-aimed manner. By nozzles 16 extending to the bottomof fermentation vessel 2, a homogeneous aerobic reaction space can beachieved. When shorter nozzles are selected, a defined space having alower oxygen partial pressure is generated. The bioreactor 1 can beoperated either without gassing (in which case the threads of pressurelid 8 can be closed by blind stoppers), or in an obligatory anaerobicmode by supplying defined gases free of O₂. In this case, any media tobe added should be degassed before being supplied.

Optionally, liquid media may also be added to bioreactor 1 from thepressure or mixing vessel 44, 46 through these nozzles 16. In this case,aeration, if required, may also be effected by the nozzle arrangement 20extending horizontally through reactor space 49.

FIGS. 4 and 5 show the nozzle arrangement 20 provided horizontallywithin fermentation vessel 2, which can be pressurized with eithercompressed air 48 or a liquid bioreactive substance 50. As can be seenfrom FIG. 5, the horizontal nozzle arrangement 20 can be supported onone side in a front wall of fermentation vessel 2 or on both sides inthe front walls. Further, the horizontal nozzle arrangement may bedesignated for support at different levels on the front wall.

As can be seen from FIG. 5, the horizontal nozzle arrangement 20consists of three pipes 24 each having a plurality of nozzle orifices 28extend horizontally in parallel through fermentation vessel 2. The threepipes 24 are interconnected through an inlet manifold 20 a and an outletmanifold 20 b.

As can be seen from FIG. 5, the horizontal nozzle arrangement 20 can berotated around a horizontal rotation axis to additionally achieve athorough mixing of the vessel contents. When the horizontal nozzlearrangement 20 is rotated, the length of pipes 14 of the vertical nozzlearrangement 10 is to be selected to preclude collision between pipes 14and pipes 24. Alternatively, if sterility of the fermentation process isnot required, the vertical nozzle arrangement may be retractedvertically upwards to such an extent that a collision between pipes 14and 24 cannot occur. To maintain sterile conditions, the rotatinghorizontal nozzle arrangement 20 is preferably provided at the lowestpossible position within fermentation vessel 2. In this case, the pipes14 are preferably shortened to the extent as just not extending into therotating area. In this case, the outermost pipes 14 which are providedclose to the sides of fermentation vessel 2 are longer than the pipesprovided further inwardly.

The outlet manifold 20 b can have a valve for closing the outlet, or itmay also be used as an inlet manifold, in which case the materialsupplied to the pipes 24 must completely pass through the nozzleorifices 28. The outlet manifold 20 b may also be completely omitted.However, this requires a more stable construction of nozzle arrangement20.

The nozzle arrangement 20 may also be used for heating/cooling byclosing the nozzles 28, or by replacing the nozzle arrangement 20 by acorresponding pipe system without nozzles. In this case, the inletmanifold 20 a serves for supplying the temperature-controlledheating/cooling fluid, and the outlet manifold 20 b serves as adischarge thereof. Such a nozzle arrangement 20 designed as a coolingarrangement may also be rotating. Further, several nozzle/coolingarrangements may be provided, so that a rotating horizontal nozzlearrangement and a rotating horizontal cooling arrangement, for example,can be provided simultaneously in one vessel 2.

Of course, it is possible to pressurize the vertical nozzle arrangement10 with compressed gas 48 and to pressurize the horizontal nozzlearrangement 20 with the bioreactive substance 50, or vice versa.

The second nozzle arrangement 20 extends horizontally through thereactor space 49. It can be Introduced at different levels and issupported to rotate around its axis, Such a nozzle arrangement is usedfor supplying the bioreactor with liquid defined media, e.g., water,required cosubstrates, vitamins, minerals, buffers or antibiotics, butmay optionally be used for aerating and mixing the bioreactor 1. For thefermentation of liquid or water-soluble substances, the horizontalnozzle arrangement may be rotated by a motor provided on the outer sideof bioreactor 1 and thus contribute to a thorough mixing of thebioreactor contents. In this case, shorter vertical pipes 14 areemployed, and the horizontal nozzle arrangement 20 is inserted in itslowermost lock.

The mixing of the bioreactor contents in the fermentation of solidsubstances and the supply of the bioreactor 1 with both atmosphericoxygen or defined gases and medium is achieved pneumatically bycompressed air. Via a pressure vessel 44, which in this case is alsoused as a media storage tank, the air gets into the pressure lid 8 andfrom there into the vertical or optionally into the horizontal nozzles16, 28. Between the pressure vessel 44 and the pressure lid 8, there isa stop valve 26. When the stop valve 26 is closed and the stop valve 7on the bottom of pressure vessel 44 is opened, medium is pressed by thecompressed air through the horizontal and optionally also through thevertical nozzle arrangement 10, 20 into bioreactor 1.

FIG. 6 shows an embodiment in which a mixing vessel 46 is connected withthe bioreactor 1 instead of pressure vessel 44. Connected to mixingvessel 46 are several pressure vessels 52, 54, 56, 58, 60 which containdifferent liquid bioreactive substances as well as compressed gas, e.g.,compressed air. These different substances can be supplied to the mixingvessel 46, where they can be mixed at a desired ratio. Through thecompressed air feed line 17 and the stop valve 18, compressed air getsto a distributing means 23 which distributes the compressed air 48 toall connected pressure vessels 52, 54, 56, 58 and 60, and to the mixingvessel 46. The compressed air feed lines 25 for the individual pressurevessels each have one stop valve 26. The compressed air line 3 for thefermentation vessel 2 also branches off from the distributing means 23.

To be able to selectively introduce different media into the presentedbioreactor 1, the pneumatic system is used from several pressure vessels52, 54, 56, 58, 60 having different volumes. FIG. 6 shows an applicationwith five exchangeable and separately autoclavable pressure vessels(e.g., for medium, two buffers, trace element solution, and antibioticssolution) in which graduated rising pipes 19 indicate the respectivefilling levels of pressure vessels 52, 54, 56, 58, 60. Through adistributing means 23, the compressed air gets into the pneumaticsystem. If needed, a part thereof can be directly introduced into thevertical and/or horizontal nozzle arrangement 10, 20 for aerating thebioreactor 1. The independently controllable pressure vessels areconnected with the distributing means 23. The respective media from theindividual pressure vessels are added to a mixing vessel 46. When themixing vessel is filled with the different media, a pressurecompensation is enabled by a pressure compensating means 47, andcompressed air, for example, is pressed into the bottom of the pressurevessel through another compressed gas line 34 of the distributing means23. This effects the mixing of the different solutions. When the mixingprocess is completed, the mixing vessel 46 is pressurized withcompressed air, and the desired medium is introduced into the horizontaland/or vertical nozzle arrangement 10, 20.

Each pressure vessel may be provided with a rising pipe 19. The feedlines 29 for the bioreactive substances from the pressure vessels eachhave a stop valve 30.

From the distributing means 23, a compressed gas line 34 furtherbranches off which contains a stop valve 35, wherein the compressed gasline 34 leads to a discharge line 37 at the bottom of the mixing vessel46 in order to supply compressed air, for example, for the mixingprocess. The discharge line is provided with a stop valve 39. From thedischarge line 37, a feed line 42 with a stop valve 43 branches throughwhich the mixed bioreactive substances 50 can be supplied to thebioreactor 1.

The mixing vessel 46 is further provided with a pressure compensatingmeans 47 for compensating the pressure during the mixing process.

The compressed gas may also be supplied in a pulsing manner to thevertical and/or horizontal nozzle arrangement 10, 20.

A measuring means 70 is connected with the fermentation vessel 2. Thismeasuring means 70 has a measuring chamber 72 which is connected withthe fermentation vessel 2 through a feed line 74. Through the feed line74, the medium to be measured flows from the fermentation vessel 2 intothe measuring chamber 72. To set the direction of flow from thefermentation vessel 2 into the measuring chamber 72 independently of theconditions in the measuring chamber 72, a check valve 76 is provided inthe feed line 74.

In the measuring chamber 72, several measuring electrodes 78 areprovided which are connected through lines 80 with a measuring means 82,which is preferably computer-controlled. The measuring electrodes 78 caneffect measurement of, for example, pH, oxygen, temperature and ions.

At the lowest position of the essentially diamond-shaped measuringchamber 72, a recirculating line 84 is connected with the measuringchamber 72. Through the recirculating line 84, the measured medium isrecirculated into the fermentation vessel 2.

To recirculate the medium into the fermentation vessel 2, pressure Isapplied to the measuring chamber 72. For this purpose, a pressurechamber 86 is assigned to the measuring chamber 72. The pressure chamber86 is connected with a source of compressed air through lines 88, 90. Toproduce a positive pressure in the pressure chamber 86, a valve 92 isopened, and a valve 94 is closed, so that the compressed air flows inthe direction of arrows 96 into the pressure chamber 86.

To recirculate medium from the measuring chamber 72 through therecirculating line 84 into the fermentation vessel 2, valve 92 isclosed, and valve 94 is opened. Thus, compressed air flows from thepressure chamber 86 in the direction of arrows 98 through a line 100connected with the measuring chamber 72 into measuring chamber 72 andthere produces a positive pressure. The defined positive pressure inpressure chamber 86 is directly proportional to the volume of liquid tobe displaced in measuring chamber 72. Due to the positive pressureproduced in measuring chamber 72, the check valve 76 in the feed line 74is closed, and the medium present in the measuring chamber 72 is pressedback through recirculating line 84 into the fermentation vessel 2. Toachieve a uniform distribution of the air pressure within the measuringchamber and to avoid eddies, a baffle 102 is provided in the region ofthe inlet of compressed air into measuring chamber 72.

Once the medium has been recirculated from the measuring chamber 72 intothe fermentation vessel 2, valve 94 is again closed, and valve 92 isopened. Thereby, a positive pressure again builds within pressurechamber 86. Once the valve 94 is closed, a compensation of pressureoccurs in the measuring chamber 72, so that the check valve 76 is againopened by medium flowing from the fermentation vessel 2. The valve 76may further be a switching valve, for example, in order to control thequantity of medium to be measured which is supplied to the measuringchamber 72.

The present invention also relates to a method for the aerobicfermentation of solid substances, wherein the reaction medium containingsaid solid substances is mixed by compressed gas 48 supplied to thereaction medium from above. According to the invention, this is effectedby introducing the compressed gas directly into the reaction mediumusing suitable means (such as a vertical nozzle arrangement 10 asdescribed above), so that the spreading gas bubbles effect mixing. Sincecompressed gas rich in oxygen (such as air, O₂-enriched air or O₂) ispreferably used in aerobic fermentation, the oxygen content in thereaction medium is also increased by this method, which usuallyaccelerates the fermentation additionally. The introducing of thecompressed gas may be effected continuously (also referred to as“aeration” in the following) or by pulses of compressed gas (alsoreferred to as “thorough mixing” in the following).

“Solid substrates” within the meaning of the method according to theinvention are preferably coal, wood and loaded soils. The methodaccording to the invention is particularly suitable for fermenting coal,especially brown coal. The latter consists of three components definedby their different solubilities as a function of pH:

1. humic acids which can be extracted by 0.1 N NaOH solution;

2. fulvic acids which are also soluble in an acidic medium;

3. the insoluble residue, referred to as the “matrix”.

For liquefying the brown coal, the starting product, which mayoptionally be pretreated or pre-oxidized, in a milled condition(particle size preferably from 0.1 mm to 2 cm, more preferably from 1 to10 mm), is mixed with an amount of solvent (i.e., water or aqueous-basedsolvent systems), nutrients, buffers (including buffer substances,acids, bases) and microorganism culture sufficient for solubilization,and cultured with thorough mixing with oxygen-containing compressed gas.

Suitable microorganisms for the solubilization of brown coal includemolds, white rot fungi and yeasts. One preferred microorganism for thisapplication is Trichoderma atroviride. The nutrients to be employed forthis fermentation method highly depend on the species of microorganismemployed. In particular, it is preferred to add carbon sources to themicroorganism which liquefies the brown coal at the beginning of thereaction to ensure a growth advantage.

Buffer substances which ensure the desired pH value at the respectivetimes of reaction are employed. Thus, at the beginning of the reaction,when a pH of from 5.5 to 6.0 is preferred, and in the liquefying phase,when a pH of from 6.5 to 7.2 is preferred, a citrate/phosphate bufferhaving a pH of 3 is preferably used, since the fungus itself alkalizesthe medium when growing, and only back titration must be performed. Itis particularly preferred to set a pH of 5.5 at the beginning of theexperiment. The fermentation is preferably performed at a temperature offrom 15 to 35° C.

In a preferred embodiment, the brown coal, the brown coal/solventmixture or the brown coal/solvent/nutrient mixture is sterilized ortyndallized prior to the addition of the fermenting microorganisms. Thisis preferably performed by several cycles of heating at temperatures ofabove 75° C., preferably above 80° C., for at least 45 minutes, followedby cooling down to room temperature for several hours.

In the 80° C. steps, the physiologically active microorganisms in thesubstrate, medium and reactor space, but not dormant spores, are killed.In the periods of time with moderate temperatures, the spores germinate,and are killed in the subsequent heating step.

Conventional tyndallization means three cycles of heating of a liquid ornutrient medium. In the intervals between the temperature steps, theproduct is kept at room temperature (Eckhard Bast, 1999,Mikrobiologische Methoden: eine Einführung in grundlegendeArbeitstechniken—Heidelberg, Berlin; Spektrum Akademischer Verlag, ISBN3-8274-0786-9).

The microbially solubilized coal obtainable according to the presentinvention can be used as a carbon and energy source for bacteria whichare capable of producing a chemically characterized substance, such aspolyhydroxyfatty acids for the synthesis of biodegradable plastics, fromthe chemically heterogeneous mass product coal (A. Steinbüchel and B.Füchtenbusch, Proceedings ICCS 97, 1673-1676 (1997)). The aliphaticresidue which cannot be liquefied microbially and has a lower proportionof water and ashes and thus a higher gross calorific value can be used,on the one hand, for direct thermal utilization (R. Köpsel et al.,Freiberger Forschungshefte, 159-166 (1998)), and on the other hand, forfurther subsequent fermentation processes by aliphatic-degrading yeasts(U. Hölker et al., Proceedings of the 16th SMYTE, Slovakia, page 16(1998); Folia Microbiol. 44, 226-227 (1999)).

It is particularly preferred to perform the method according to theinvention in the above described bioreactor.

The present invention is further illustrated by the following Example.

EXAMPLES General procedures

The product “liquefied coal” was defined as the sample supernatantobtained after 20 min of centrifuging at 10,000×g. The degree ofliquefaction was determined from the optical density at 450 nm or fromthe dry weight of the supernatant. To separate the humic acid compoundsfrom fulvic acid compounds, the supernatant was acidified to pH 1.5 andagain centrifuged. The liquefied products were characterized withrespect to their optical densities, humic and fulvic acid contents andbacterial contamination by means of incubation of samples in completemedia followed by microscopic analyses.

Example 1 Fermentation of Brown Coal

A bioreactor according to the invention (as shown in FIGS. 1 to 6 havinga vertical nozzle arrangement (10), a pressure lid (8) with pipes (14)having nozzles (16) and extending to the bottom of the reactor space(49), and a rigid horizontal nozzle arrangement (20); volume; 25 l) wascharged with 2,500 g of brown coal (Bergheim Lithotyp A, particle sizefrom 2 to 10 mm, water content of the coal about 50%) as a solid to beconverted in 10 l of water. As the coal-solubilizing aerobic fungus,Trichoderma atroviride was employed (U. Hölker et al., Fuel ProcessingTechnol., 52, 65-71 (1997)). Fifty grams of glutamate was added toinitiate the induction of coal-liquefying enzymes (U. Hölker et al.,Appl. Microbiol. Biotechnol. 44, 351-255 (1995)). The initial pH was 5.8in order to provide the fungus with a growth advantage at first over thebacteria present in the coal. The continuous air pressure sufficient foraeration was 0.4 bar. The air pressure was increased to 3 bar for 10 sdaily by the vertical nozzle arrangement 10 in order to thoroughly mixthe reactor contents.

In a semicontinuous approach, 800 ml of water was added through thehorizontal nozzle arrangement (20) at intervals of 24 hours, and thesame quantity of reactor contents was withdrawn at the discharge system.In this suspension, the optical density, pH, humic and fulvic acidcontents were determined and checked for bacterial contamination. If thepH value reached 7.3, it was titrated back to 7.0 via the media supplysystem (FIG. 8, arrows 1-5).

The recovery of solubilized coal sought in this fermentation approachwas 3 mg of dry weight per ml of suspension at pH 7 per day and shouldbe kept constant continuously over a period of 30 days. This correspondsto a sought recovery of about 2 g of solubilization products per day.When the recovery increased beyond 3.3 mg of dry mass per ml, thereactor contents were diluted by adding water through the media supplysystem and again adjusted to the desired concentration (FIG. 8, arrows6-10).

After a fermentation period of 12 days, the sought concentration ofsolubilized coal was reached, and within a period of another 28 days, 71g of solubilization products was produced in the proposed bioreactor.

Example 2 Method for the Mild Sterilization of Bioreactors withContents; Modified Tyndallization

A bioreactor according to the invention (as shown in FIGS. 1 to 6,having an insertable vertical nozzle arrangement (10) with short pipes(14), devices for heat exchange (51 a, 51 b, 51 c) in the outer wall ofa rotating horizontal nozzle arrangement (20) and a measuring means(70); volume: 12.5 l-) was charged exactly as described in Example 1.The pressure lid (8) was closed and the nozzles (16) were thuspneumatically pressed into the substrate. The pneumatic recirculatingand measuring system was activated, and at one-minute intervals, themedium was pumped past the temperature sensor of the measuring chamberand through the horizontal nozzle system (20) back into the bioreactor.Water was heated in a thermostat at 95° C. and pumped through the jacketof the bioreactor (51 a, 51 b, 51 c) until the temperature in theinterior reached 80° C. This temperature was maintained for 45 min (withconstant aeration/thorough mixing by the vertical nozzle system (10) andrecirculating of the medium through the horizontal nozzle system (20)).Subsequently, the bioreactor was cooled down to 25° C. bytemperature-controlled water in the jacket, while it was aerated,thoroughly mixed and recirculated for 12 hours. Subsequently, thetemperature in the bioreactor space was again adjusted to 80° C. for 45minutes as before. This was again followed by cooling down to 25° C. for20 hours, and then a third heating of the reactor interior to 80° C. for45 min.

To verify that a successful sterilization had taken place, thebioreactor was subsequently further thoroughly mixed, aerated andrecirculated. Daily (for 7 days), 1 ml samples were taken and used toinoculate both Petri dishes (1.2% agar) and 50 ml liquid culturescontaining the nutrient medium used in the bioreactor. The Petri disheswere incubated at 25° C. for 72 hours, and the liquid cultures wereincubated at 255° C. and 120 rpm on a vibrator. It was found that nocontaminations could be detected under such conditions in the reactorspace.

After the sterility control had been performed, the bioreactor wasinoculated with the coal-solubilizing fungus T. atroviride, and the coalwas fermented by analogy with Example 1, but at a set temperature of 25°C. to obtain 60 g of liquefied product.

What is claimed is:
 1. A bioreactor for fermenting solid substrates,comprising a fermentation vessel, a charging means for bioreactivesubstances, and at least one nozzle arrangement within said fermentationvessel for aeration and thorough mixing of the substrates, wherein theat least one nozzle arrangement has a multitude of pipes extending inparallel into a reaction space of the fermentation vessel and providedwith nozzles, wherein a first, vertically extending nozzle arrangementcan be extended into and retracted from the reaction space of thefermentation vessel, and having a second, horizontal nozzle arrangementwith at least one pipe having nozzle orifices and extending horizontallythrough the reaction space.
 2. The bioreactor according to claim 1,wherein a second, horizontal nozzle arrangement is provided whichconsists of at least two interconnected pipes extending horizontally andin parallel through the reaction space, each having a plurality ofnozzle orifices.
 3. The bioreactor according to claim 1, wherein saidhorizontal nozzle arrangement can be rotated around a horizontalrotation axis.
 4. The bioreactor according to claim 1, wherein saidfermentation vessel has a bottom section with a tapering cross-section.5. The bioreactor according to claim 4, wherein said bottom section isconically designed and leads into a draining channel which is inclinedfrom horizontal and has a draining valve at the lowest position thereof.6. The bioreactor according to claim 1, wherein said at least one nozzlearrangement receives compressed gas from a pressure vessel.
 7. Thebioreactor according to claim 6, wherein said pressure vessel contains abioreactive liquid substances, wherein said bioreactive liquid substanceis brown coal suspended in a liquid and the liquid has a pH in range of5-7 pH unit in addition to said compressed gas.
 8. The bioreactoraccording to claim 7, wherein said at least one nozzle arrangementalternatively receives compressed air or said liquid bioreactivesubstance from said pressure vessel.
 9. The bioreactor according toclaim 1, wherein said at least one nozzle arrangement can be pressurizedwith pulsing compressed air.
 10. The bioreactor according to claim 1,wherein said second nozzle arrangement is provided within saidfermentation vessel in a height-adjustable manner.
 11. The bioreactoraccording to claim 7, wherein a multitude of pressure vesselspressurized with compressed air and connected to a mixing vessel areprovided which contain different liquid bioreactive substances.
 12. Thebioreactor according to claim 11, wherein said mixing vessel has apressure compensating means.
 13. The bioreactor according to claim 11,wherein said pressure vessels are exchangeable and can be separatelyautoclaved.
 14. The bioreactor according to claim 5, wherein saiddraining channel is covered by a wire mesh.
 15. The bioreactor accordingto claim 1, wherein a pressure lid of said fermentation vesselaccommodates said first nozzle arrangement whose pipes extend verticallyfrom the pressure lid into the reaction space.
 16. The bioreactoraccording to claim 15, wherein said vertical pipes of said first nozzlearrangement are provided in said pressure lid to be exchangeable. 17.The bioreactor according to claim 1, wherein said fermentation vessel isconnected through a feed line with a measuring chamber, which is againconnected through a recirculating line with said fermentation vessel,and said measuring chamber can be pressurized for recirculating measuredmedia.
 18. The bioreactor according to claim 1, wherein a device forheat exchange is provided comprising a device: (i) in which saidfermentation vessel has a double wall and the thus formed cavity can beflowed through with temperature-controlled heat exchange fluids througha connecting pipe and discharge pipe; and/or (ii) which is a horizontalpipe system within said fermentation vessel which can be flowed troughwith a temperature-controlled heat exchange fluid.
 19. A method ofproducing a fermentation product, said method comprising the aerobicfermentation of a reaction medium comprising solid substrates, whereinsaid reaction medium comprises brown coal, culture medium, and amicroorganism capable to ferment said solid substrate to produce afermentation product, and the recovery of the fermentation productproduced, wherein said reaction medium containing the solid substratesis thoroughly mixed by compressed gas supplied to said reaction mediumfrom above.
 20. The method according to claim 19, wherein said thoroughmixing is effected by a continuous stream of compressed gas or bycompressed gas pulses.
 21. The method according to claim 19, whereinsaid solid substrates are selected from the group consisting of coal,wood and loaded soils.
 22. The method according to claim 21, whereinsaid solid sub is coal.
 23. The method according to claim 22, whereinthe solid substrate is brown coal also known as lignite.
 24. The methodaccording to claim 22, which further comprises adding a microorganismcapable to ferment said solid substrate, nutrients and/or buffers tosaid reaction medium.
 25. The method according to claim 23, wherein,said brown coal or the reaction medium containing said brown coal istyndallized together with a bioreactor prior to fermentation or prior tothe addition of a microorganism.
 26. The method according to claim 23,wherein: (i) said brown coal has a particle size of from 1 to 10 mm;(ii) a microorganism is added to the reaction medium, and themicroorganism is selected from the group consisting of molds, yeasts andwhite rot fungi; (iii) the pH of the reaction medium is from 5.5 to 6.0at a beginning of the reaction; (iv) the pH is maintained at from 6.5 to7.2 during a solubilization phase; (v) the fermentation is performed ata temperature of from 25° C. to 30° C.; and/or (vi) from 1 to 25 literof compressed air per liter of fermentation broth per day is passedthrough the reaction medium.
 27. The method according to claim 26,wherein the microorganism added to said reaction medium is Trichodermaatroviride.
 28. The method according to claim 19, wherein saidfermentation is performed in a bioreactor comprising a fermentationvessel, a charging means for said reaction medium, and at least onenozzle arrangement within said fermentation vessel for aeration andthorough mixing of the substrates, wherein the at least one nozzlearrangement has a multitude of pipes extending in parallel into areaction space of the fermentation vessel and provided with nozzles,wherein a first, vertically extending nozzle arrangement can be extendedinto and retracted from the reaction space of the fermentation vessel,and having a second, horizontal nozzle arrangement with at least onepipe having nozzle orifices and extending horizontally through thereaction space.